Many differentiated but renewable cell types, including blood, skin, and sperm, are derived from dedicated precursor cells, or stem cells, which maintain the essentially unlimited capacity for continued division. Despite their biological and medical importance, the mechanisms that control stem cell behavior are poorly understood. We propose to identify molecular mechanisms that specify stem cell identity, self-renewal, and commitment to differentiation, using the Drosophila germ line as a model tissue. We have identified three genes required for normal germ line stem cell behavior and fate: zonder kloten (zk) acts in establishment of male germ line stem cells, one shot (osho) is required for male germ line stem cell self-renewal, and stem cell tumor (stet) is required in both sexes for commitment to or initiation of differentiation by stem cell daughters. To pinpoint at which step(s) in stem cell establishment and self-renewal the genes act, we will examine embryonic and adult gonads for the earliest defects in the mutants using a variety of available markers, plus double mutant analysis. To elucidate the mechanism of action of these genes in specifying tissue renewal from dedicated stem cells, we will construct germ line/soma chimeras by pole cell transplantation to determine whether zk, osho, and stet function in the germ line itself or in somatic support cells and we will identify the zk, osho, and stet transcription units, sequence corresponding cDNAs, and examine the predicted protein products for structural motifs and homology to proteins of known function. Finally, we will use powerful first generation genetic selections for suppressors of osho to identify additional genes in the pathway of stem cell self- renewal. Variations of the mechanisms we uncover may govern stem cell behavior in other organisms and tissues, leading to applications in cancer, therapeutics, and genetic engineering.